Role Of Mitochondrial Calcium Uniporter In Early Brain Injury After Xperimental Subarachnoid Hemorrhage

Background:Spontaneous subarachnoid hemorrhage(SAH),usually caused by cerebral aneurysm rupturing,is a frequently seen devastating disease.With its high mortality and disability,SAH imposes a heavy burden on society and economy.Past studies have focused primarily on cerebral vasospasm(CVS),and the reduction in angiographic vasospasm did not translate into a measurable clinical benefit in a clinical trial.Recent studies have indicated that the pathophysiological event occurring within 72 h after SAH,as termed as early brain injury(EBI),is the most important factor determining the prognosis of patients suffering from SAH.Various mechanisms have been attributed to the pathogenesis of EBI after SAH,including oxidative stress,impaired calcium homeostasis,iron accumulation,inflammation and apoptosis.Mitochondrial calcium uniporter(MCU)locates in the inner membrane of mitochondria.It can be inhibited by ruthenium red(RR)and opened by polyamine like spermine(Sper).Under physiological conditions,mitochondrial calcium uptake is undertaken by MCU.Cellular calcium overload and iron accumulation have been proved invloved in the acute phase following SAH.Treatment of nimodipine,a calciumchannel blocker,can improve neurological outcomes but not cerebral vasospasm in clinical tests.Also,in animal experiments,systemic administration of deferoxamine,an iron chelator,could reduce iron level and iron-handling proteins,and significantly ameliorated oxidative injury and neuronal cell death.Cellular and hence mitochondrial calcium accumulation,associated with increased reactive oxygen species(ROS),mediate the sustained opening of mitochondrial permeability transition pore(mPTP).As a result,mitochondrial pro-apoptotic proteins,such as cytochrome c(cyt c)are released from the mitochondria to the cytoplasm.In addition,owing to iron’s ability to donate electrons to oxygen,increased iron levels can lead to the formation of hydroxyl radicals and hydroxyl anions via the Fenton Reaction,thus aggravating brain injury following SAH.Previous studies have shown that mitochondrial calcium/iron accumulation is associated with the physiopathologic process of many diseases such as the ischemia/reperfusion injury or iron overload associated injury both in brain and heart.Nesides,blockage of MCU could prevent mitochondrial dysfunction in these models.However,the exact molecular mechanism of the role of calcium after SAH has not been fully elucidated.The purpose of this study is to clarify the subcellular calcium overload and iron accumulation and to investigate the potential role of MCU as a therapeutic target in EBI following SAH.Objectives:The aims of this study are:1)to establish a stable SAH model in rat;2)to investigate the protective effect of blockage of MCU on EBI following SAH;3)to explore the effect of blockage of MCU on iron accumulation following SAH.Methods:Male Sprague-Dawley(SD)rats were randomly divided into the sham(surgery with normal saline insult),SAH,SAH+RR(SAH treated with RR 2.5 mg/kg),and SAH+Sper(SAH treated with Sper 5 mg/kg)groups.RR or Sper(both from Sigma,dissolved in sterile saline solution)were injected directly intraperitoneally(IP)at 15 min after SAH.Before execution,the neurological evaluation was finished.Biochemical analysis,including measurement of adenosine triphosphate(ATP),reactive oxygen species(ROS),cellular/mitochondrial Ca2+ levels,aconitase activity and western blot,and histological assays including TUNEL,iron and Nissl staining were analyzed.Results:The data obtained with biochemical and histological assays showed that mitochondrial Ca2+ concentration was significantly increased in the temporal cortex of rats 1,2,and 3 days after SAH,consistent with constant high levels of cellular Ca2+concentration.In agreement with the observation in the acute phase,SAH rats showed an obvious increase of ROS level and decrease of ATP production.Blockage of MCU prevented Ca2+ accumulation,abated the level of oxidative stress,aUd improved the energy supply.Translocation of cytochrome c,increased cleaved caspase-3,and a large amount of apoptotic cells after SAH were reversed by RR administration.Surprisingly,exogenous spermine did not increase cellular Ca2+ concentration,but lessened the Ca2+ accumulation after SAH to benefit the rats.Besides The results confirmed the iron accumulation in temporal lobe after SAH.Blockage of MCU dramatically reduced the iron accumulation in this area.The mechanism was revealed that inhibition of MCU reversed the down-regulation of iron regulatory protein(IRP)1/2 and increase of ferritin.Iron-sulfur cluster dependent-aconitase activity was partially conserved when MCU was blocked.Conclusion:In the acute phase after SAH,mitochondrial calcium overload and iron accumulation may produce a large amount of ROS and lead to cellular bioenergetic crisis,which in turn contributed to cell death.Blockage of MCU could notably alleviate calcium overload and iron accumulation,which in turn could significantly ameliorate oxidative injury and reduce neuronal cell death.Thus,MCU may be a potential therapeutic target for patients suffering from SAH.